Synthetic spider silk proteins and expression thereof in transgenic plants

ABSTRACT

The invention relates to a DNA sequence coding for a synthetic protein and recombinant spider silk proteins which are coded by the inventive DNA sequence. The invention also relates to methods for producing plants or plant cells containing the recombinant spider silk protein and transgenic plants and cells containing a DNA sequence coding for a synthetic spider protein. The invention further relates to a method for obtaining a vegetable spider silk protein from transgenic plants in addition to vegetable spider silk proteins produced according to said method.

RELATED APPLICATIONS

This is the national phase of PCT/EPO/06586 filed Jun. 11, 2001, whichclaim priority to DE 100 28 212.1 filed Jun. 9, 2000, DE 100 53 478.3filed Oct. 24, 2000 and DE 101 13 781.8 filed Mar. 21, 2001, the entirecontents of which are incorporated.

The invention relates to a DNA sequence that codes for a syntheticspider silk protein, recombinant spider silk proteins coded by the DNAsequence according to the invention, methods of producing plants orplant cells containing recombinant spider silk protein, as well astransgenic plant cells and plants containing a DNA sequence that codesfor a synthetic spider silk protein. In addition, the invention relatesto a method of obtaining plant spider silk protein from transgenicplants, as well as plant spider silk proteins produced according to saidmethod.

Spider silk exhibits outstanding mechanical properties that are superiorto those of many known natural and synthetic materials. The mainconstituents of spider silk are fibre proteins, e.g., fibroin, from thesilkworm, as well as spidroin 1 and spidroin 2 from Nephila clavipes.The strength and elasticity of the silk are based on the presence ofshort, repetitive amino acid units within these natural proteins. Thesemechanical properties predestine the spider silk for a series of themost varied technical applications, e.g., the manufacture of stablethreads or silks. In addition, due to their protein chemical propertiesthe spider silk threads have a low immunogenic and allergenic potential,so that, when combined with their mechanical

properties, these threads can be beneficially used in medicine, e.g., asa natural yarn for closing wounds, as adhesion surfaces for cultivatedcells, as frames for artificial organs and the like.

However, one prerequisite for such technical or medical use of thespider silk is the large-scale production of spider threads or spidersilk proteins. To this end, attempts have been made up to now to expressthe spidroin or fibroin genes responsible for the production of thespider silk in E. coli. However, during reproduction in bacteria thefrequently repeated sequences in the corresponding genes are graduallylost. Another problem is the quantity of genetic information, whichappears to be too extensive for the bacterium, so that a completereadout of the spider silk genes is not always possible.

While expression experiments in yeast cells yielded more stable andlonger silk proteins, the threads spun from them do not exhibit the sameadvantageous properties of natural silk, so that such syntheticallyproduced silk cannot be used for example for medical purposes. There isthus a need for synthetic silk proteins that can be produced on anindustrial scale which after spinning into threads display mechanicalproperties comparable with those of natural silk.

Therefore, the object of the present invention is to provide DNAsequences that code for a synthetic spider silk protein as similar aspossible to the previously known natural sequences of fibre proteins inspider silk. In addition, the object of this invention is to provide amethod according to which synthetic spider silk proteins can be producedon a large-scale.

The object of the invention is also to provide DNA sequences that codefor a synthetic spider silk protein exhibiting the advantageous anddesirable properties of native spider silk protein, but where the rangeof properties of the native protein has additionally been modified oroptimised in this way or that, depending on the intended application.

Other objects of this invention will become clear from the followingdescription.

The above objects are achieved by the features in the independentclaims.

Advantageous embodiments are described in the sub-claims.

The DNA sequence disclosed by the present invention codes for asynthetic fibre protein, in particular a synthetic spider silk proteinexhibiting a homology of at least 80%, preferably of at least 84%, morepreferably of at least 88%, especially preferably of at least 90% and92%, and most preferably of at least 94% with spidroin and/or fibroinproteins, in particular with the spidroin 1 protein, especiallypreferably with the spidroin 1 protein from Nephila clavipes.

Within the context of this invention, homology denotes similaritybetween amino acid sequences based on identical or homologous amino acidstructural units. The person skilled in the art knows which amino acidsare to be regarded as homologous, e.g., (i) isoleucine, leucine andvaline among each other, (ii) asparagine and glutamine, (iii) asparticacid and glutamic acid.

The DNA sequence according to the invention is composed of modulescomprising a group of successively arranged oligonucleotide sequences,wherein the oligonucleotide sequences each

code for repetitive units from spidroin and/or fibroin proteins.

The structure of the inventive DNA sequence composed of various modules,which are in turn made out of different short amino acid repeats typicalfor spidroins or fibroins, whereby the principle of successivelyarranging the corresponding oligonucleotide sequences or modules isoriented towards natural spidroin and/or fibroin sequences, ensures avery high homology to previously known natural spidroin or fibroinsequences. This ensures that the spider silk proteins coded by the DNAsequence according to the invention after being spun into threads willexhibit outstanding mechanical properties in terms of their strength andelasticity, which are comparable to the mechanical properties of naturalspider threads.

In addition, the modular structure of the DNA sequence according to theinvention makes it possible to modify the synthetic genes quite simplyby means of genetic engineering, so that multimers of synthetic spidersilk proteins of any size can be produced as desired. Further, thespider silk proteins coded by the DNA sequence according to theinvention can, due to their modular structure, be fused with other fibreprotein sequences. One special advantage of the DNA sequence of thepresent invention is that due to its modular structure it is easy tofuse with sequences that code for purifying elements orsolubility-altering peptides.

The invention also relates to DNA sequences that code for a syntheticspider silk protein and which are comprised of modules comprising agroup of successively arranged oligonucleotide sequences, whereby eachof the oligonucleotide sequences codes for repetitive units fromspidroin proteins and the modules are freely arranged, the freearrangement making it possible for synthetic spider silk protein toexhibit an altered range of properties compared to native spider silkprotein.

Therefore, the invention makes it possible, for the first time, tosynthesize new types of silk proteins based on modular structured silkprotein genes, the new types of silk proteins having a modified range ofproperties compared to native silk protein, while at the same timecontaining the essential structural determinants of naturally occurringsilk proteins. While maintaining the essential structural sections ofnatural silk proteins, which are combined with each other in a novelmanner according to the invention, synthetic silk proteins are providedwhich, with regard to their elasticity, tensile strength, solubilitybehaviour, heat and acid resistance and swelling capacity, are modifiedor optimised in a particular way depending on the particular purpose.

Specific arrangements of the obtained synthetic proteins can make theobtained protein particularly well suited for a specific purpose. As analternative, of course, one can screen for a protein particularly suitedfor a specific application, e.g. having increased elasticity compared tonative protein. Increased elasticity may be achieved by purposely usingmore elastic modules for the structure instead of rigid modules.

In any event, the combination of properties, which makes the recombinantspider silk proteins according to the invention so useful and attractivefrom a material/technical point of view, can be influenced withindesired limits by the arrangement of the modules, without differing toomuch from the attractive range of properties of the natural protein.

The gene cassette with the highest homology to the cDNA isolated fromthe native host, called SO1, exhibits the following combination ofstructural sections designated as a module (represented by variousletters): H_B_C_B_C_G_D_C_G_D_C_B_C_B_B_G_D_B_C(see also FIG. 3). In contrast to the approaches in the prior art withrespect to spider silks and natural silks, the teaching of the presentinvention for assembling the gene cassettes allows a new and targetedarrangement of these modules in a completely variable manner. This makesit possible to create completely new types of proteins, and also toreconstruct the naturally occurring protein. In addition to the modulesequence series shown above for the naturally occurring sequence, anynumber of variations in any scheme are thus now possible, such as thefollowing, each of which yield proteins having different properties:H_(n)≠B_(n)≠C_(n)≠D_(n)≠(H_(x)B_(y))_(n)≠(H_(x)C_(y))_(n)≠ . . .≠(H_(i)B_(j)C_(k)D_(l))_(n).Embodiments for the possibilities of creating such structures and forthe different properties of the resulting proteins can be gathered fromthe examples provided below.

In addition to the properties already mentioned, which can be furthermodified or optimised, additional RGD sequences, for example, may beused to achieve an enhanced adhesion of cells (Massia et al. (2001), J.Biomed. Mater. Res. 56: 390-399). Other useful properties of thesynthetic spider silk proteins according to the invention also may bederived from the following description and examples.

In a particularly preferred embodiment of this invention, the spidersilk protein coded by the DNA sequence according to the invention has ahomology of at least 84%, preferably of at least 90%, and especiallypreferably of at least 94% with the spidroin 1 protein from Nephilaclavipes. Spidroin 1 from Nephila clavipes is significantly involved inthe structure of a support thread that is mechanically particularlystable and elastic.

The modular structure of the DNA sequence according to the inventionrenders it possible to construct genes that encode very large spidersilk proteins, wherein the high degree in homology with spidroin and/orfibroin proteins, in particular with spidroin 1, especially preferablywith spidroin 1 from Nephila clavipes, is always retained. The sizedistribution achievable in this way for the proteins coded by the DNAsequences according to the invention corresponds to the range of spidersilk proteins that can be observed after dissolving natural spider silk.This identical range of sizes as well the high sequence homology definesthe synthetic genes according to the invention as genes that code forspider silk proteins. In contrast to natural spider silk, which consistsof a mixture of spider silk proteins, this invention provides spidersilk protein genes that represent a gene class by having high homology,and permit simple gene-technological manipulation.

The modules for assembling the DNA sequence of the present inventioncomprise a group of successively arranged oligonucleotide sequences,which preferably are selected from the group consisting of: (SEQ IDNO: 1) a) TATGAGCGCTCCCGGGCAGGGT; (SEQ ID NO: 2) b)AGCTTTTAGGTACCAATATTAATCTGGCCGGCTCCACC; (SEQ ID NO: 3) c) TATGGTCTGGGG;(SEQ ID NO: 4) d) GGCCAGGGTGCTGGCCAA; (SEQ ID NO: 5) e)GGTGCAGGAGCWGCWGCWGCWGCTGCAGGTGGA; (SEQ ID NO: 6) f)GCCGGCCAGATTAATATTGGTACCTAAA; (SEQ ID NO: 7) g) CTGCCCGGGAGCGCTCA; (SEQID NO: 8) h) ACCACCATAACCTCC; (SEQ ID NO: 9) i) AGCACCCTGGCCCCCCAG; (SEQID NO: 10) j) TGCAGCWGCWGCWGCWGCTCCTGCACCTTGGCC; (SEQ ID NO: 11) k)TATGAGATCTGGCCAAGGAGGT; (SEQ ID NO: 12) l) TTGGCCAGATCTCA; (SEQ ID NO:13) m) AGTCAGGGTGCTGGTCGTGGAGGCCAA; (SEQ ID NO: 14) n)TCCACGACCAGCACCCTGACTCCCCAG; (SEQ ID NO: 15) o)AGTCAGGGCGCTGGTCGTGGGGGACTGGGTGGCCAA; (SEQ ID NO: 16) p)ACCCAGTCCCCCACGACCAGCGCCCTGACTCCCCAG; (SEQ ID NO: 17) q)CTGGGAGGGCAGGGAGCGGGCCAA; (SEQ ID NO: 18) r) CGCTCCCTGCCCTCCCAGACCTCC;and s) sequences that exhibit at least 80%, preferably at least 90%,especially preferably at least 94% sequence identity to the sequences ofa) to r).

The modules preferably comprise at least four oligonucleotide sequences,which preferably differ, in order to mimic the natural spider silkproteins in an authentic manner. The DNA sequence according to theinvention in turn is preferably composed of at least four of the modulesdescribed above.

The structure of the DNA sequence according to the invention isdescribed below by way of example. First of all, the oligonucleotidesshown in FIG. 1 are prepared, which code for amino acid sequencescorresponding to spidroin-typical, short amino acid repeats. Theseoligonucleotides are combined with each other using gene technologicalmethods, the combination being geared towards the natural spidroinsequence (see FIG. 2). Modules A, B, C, D, E and F obtained in this wayare again combined with each other (see FIG. 3). In this way, DNAsequences according to the invention are provided, which exhibit ahomology of at least 85%, preferably of at least 90%, and particularlypreferably of at least 94% with spidroin proteins at the amino acidlevel.

In a further embodiment, the DNA sequence according to the inventioncomprises in addition to the modules described above nucleic acidsequences that code for repeated units from fibroin proteins, preferablyfrom the fibroin protein of the silkworm.

Sequences SEQ ID NO: 19 to 29 exhibit especially preferred DNA sequencesaccording to the invention.

In addition, the invention has surprisingly succeeded for the first timein creating synthetic spider silk proteins in transgenic plants. In thisway, synthetic spider silk proteins can be produced on a large scale. Toensure stable expression of the DNA sequence according to the inventionin plants, a recombinant nucleic acid molecule is provided thatcomprises the DNA sequence according to the invention described above,as well as an ubiquitously acting promoter, preferably the CaMV 35Spromoter. The provision of the recombinant nucleic acid moleculeaccording to the invention permits the expression and accumulation ofsynthetic spidroin or fibroin sequences in transgenic plants.

To ensure that the DNA sequence according to the invention is expressedand accumulated in suitable compartments of transgenic plants, thenucleic acid molecule according to the invention comprises, in additionto the DNA sequence according to the invention and the ubiquitouslyacting promoter, preferably at least one nucleic acid sequence thatcodes for a plant signal peptide.

In a preferred embodiment, the endoplasmatic reticulum (ER) is theselected compartment for the expression or accumulation of the syntheticspider silk protein. This compartment is particularly suitable forstable the accumulation of foreign proteins in plants. To ensuretransport into the ER, the nucleic acid molecule according to theinvention preferably comprises corresponding signal peptides, the LeB4Spsequence being particularly preferred.

ER retention, if desired, is ensured according to the invention in thatthe nucleic acid molecule according to the invention additionallycomprises a nucleic acid sequence coding for an ER retention peptide.Retention in the ER is preferably achieved by the amino acid sequenceKDEL (SEQ ID NO: 52) attached to the C terminus.

In addition, it may be advantageous to place the DNA sequence accordingto the invention at the plasmalemma, i.e., the cell membrane. For thisreason, in an alternative embodiment the recombinant nucleic acidmolecule according to the invention comprises the DNA sequence accordingto the invention fused with the N terminus of a transmembrane domain.Preferably, this transmembrane domain is the transmembrane domain of thePDGF receptor, the so-called HOOK sequence (see FIG. 4).

In a especially preferred embodiment of this invention, the nucleic acidmolecule according to the invention is fused with ELPs (elastin-likepolypeptides). ELPs are oligomeric repeats of the pentapeptideVal-Pro-Gly-Xaa-Gly ((SEQ ID NO: 53), wherein Xaa is every amino acidexcept proline and is preferably Gly), and are subjected to a reversibleinverse temperature transition. They are very soluble in water below theinverse transition temperature (T₁), but have a sharp phase transitionstate in the range of 2° C. to 3° C., when the temperature is increasedto above T₁, which leads to precipitation and aggregation of thepolypeptide. D. E. Meyer and A. Chilkoti, Nat. Biotech. 1999, 17:1112-1115, have described that ELP fusions with recombinant proteinsalter the solubility behaviour of these recombinant proteins at varioustemperatures and concentrations in a targeted fashion. In the presentinvention, this is used to establish purification strategies describedin detail below for the spider silk protein coded by the DNA sequenceaccording to the invention. Preferably, the ELPs coded by the nucleicacid sequence in the nucleic acid molecule according to the inventioncomprise from 10 to 100 of the pentameric units described above (seeFIG. 5).

The chimeric gene constructs or recombinant nucleic acid moleculesdescribed above are produced using conventional cloning techniques (seefor example Sambrook et al. (1989), Molecular Cloning: A LaboratoryManual, 2^(nd) edition, Cold Spring Harbour Laboratory Press, ColdSpring Harbour, New York). These typical molecular biological techniquesmake it possible to prepare or produce desired constructs for thetransformation of plants. Methods for cloning, mutagenesis, sequenceanalysis, restriction analysis and other additionalbiochemical/molecular biological methods commonly used for genetechnologically manipulating prokaryotic cells are well known to theperson skilled in the art. Thus, it is not only possible to producesuitable chimeric gene constructs containing the respectively desiredfusion of promoters, DNA sequence according to the invention, sequencecoding for a plant signal peptide, sequence coding for an ER retentionpeptide, sequence coding for a transmembrane domain and/or sequencescoding for purifying elements or solubility-altering peptides, butrather the person skilled in the art may use routine techniques tointroduce various mutations or deletions into the respective genes, ifdesired.

The invention also relates to vectors and microorganisms that containnucleic acid molecules according to the invention, and whose use renderspossible the production of plant cells or plants that produce spidersilk proteins. These vectors include in particular plasmids, cosmids,viruses, bacteriophages and other vectors common in genetic engineering.The microorganisms are primarily bacteria, viruses, fungi, yeasts andalgae.

Since the DNA sequences according to the invention, because of theirrepetitive nature, exhibit hardly any unique restriction sites, thevectors according to the invention or the genes encoding the syntheticspider silk protein were adapted accordingly using various strategies(see FIGS. 6 to 8). When the DNA sequences according to the inventionare amplified by PCR, preferably oligonucleotides are first ligatedthereto due to the extremely repetitive nature of the DNA sequencesaccording to the invention, which then serve as templates for thesubsequent PCR reactions (see FIG. 7).

Furthermore, the present invention provides a recombinant spider silkprotein that is coded by the DNA sequence according to the invention.This synthetic spider silk protein according to the invention,preferably having a molecular weight ranging from 10 to 160 kDa,exhibits a homology of at least 85%, preferably of at least 90%, andparticularly preferably of at least 94% with spidroin and/or fibroinproteins. This high degree of homology with the natural fibre proteinsof the spider and silkworm ensures that the outstanding mechanicalproperties of the natural spider threads are achieved when the proteinsaccording to the invention are spun into threads.

In addition, the proteins according to the invention surprisinglyexhibit novel physicochemical properties. For example, the solubility ofthese synthetic fibre proteins according to the invention is sustainedextremely well in aqueous solutions, even after prolonged boiling. Inconjunction with the also occurring solubility in organic solutions andthe precipitation behaviour in the presence of high salt concentrations,these new properties of the synthetic spider silk proteins according tothe invention may therefore be used to develop technically feasibleextraction and purification techniques. These properties are enhancedeven further if the synthetic spider silk proteins according to theinvention are specifically accumulated in specific compartments, inparticular in the ER of transgenic plants.

Examples of amino acid sequences of the recombinant synthetic spidersilk proteins according to the invention are the sequences identified inSEQ ID NO: 30 to 40. Alternatively, the spider silk proteins accordingto the invention may also be synthesized according to chemical methodsknown to the person skilled in the art, although recombinant manufactureis preferred.

The invention also relates to a method for manufacturing spider silkprotein-producing plants or plant cells, comprising the following steps:

-   a) Manufacture of a recombinant nucleic acid molecule according to    the invention as described above,-   b) Transfer of the nucleic acid molecule from a) to plant cells; and-   c) optionally, regeneration of fertile plants from the transformed    plant cells.

In addition, the invention relates to plant cells containing the nucleicacid molecules according to the invention or the vector according to theinvention. The invention also concerns harvest products and propagatingmaterial of transgenic plants, as well as the transgenic plants thereof,which contain a nucleic acid molecule according to the invention.

To prepare the introduction of foreign genes into higher plants, ortheir cells, a large number of cloning vectors are available whichcontain a replicating signal for E. coli and a marker gene for selectingtransformed bacterial cells. Examples of such vectors are pBR322, pUCseries, M13 mp series, pACYC184 etc. The desired sequence may beintroduced into the vector at a suitable restriction site. The resultingplasmid is then used for the transformation of E. coli cells.Transformed E. coli cells are cultivated in a suitable medium and thenharvested and lysed, and the plasmid is recovered. The analytic methodsused to characterise the produced plasmid DNA generally includerestriction analyses, gel electrophoreses and other biochemical andmolecular biological methods. After each manipulation step the plasmidDNA may be cleaved and the obtained DNA fragments may be linked to otherDNA sequences.

A plurality of techniques is available for introducing DNA into a planthost cell, and the person skilled in the art will not have anydifficulties in selecting a suitable method in each case. Thesetechniques comprise the transformation of plant cells with T-DNA by useof Agrobacterium tumefaciens or Agrobacterium rhizogenes as thetransforming agent, the fusion of protoplasts, injection,electroporation, the direct gene transfer of isolated DNA intoprotoplasts, the introduction of DNA by means of biolistic methods aswell other possibilities that have been well established for severalyears and belong to the normal repertoire of the person skilled in theart of plant molecular biology or plant bioengineering.

For injection and electroporation of DNA in plant cells, no specialrequirements are imposed per se on the used plasmids. The same appliesto direct gene transfer. Simple plasmids, such as pUC derivatives can beused. However, if entire plants are to be regenerated from thesetransformed cells, the presence of a selectable marker gene isrecommended. The person skilled in the art is familiar with currentselection markers, and he would have no problem choosing a suitablemarker.

Depending on the method for introducing desired genes into the plantcell, additional DNA sequences may be required. If, for example, the Tior Ri plasmid is used for the transformation of the plant cell, at leastthe right border, however more often both the right and left border ofthe T-DNA contained in the Ti or Ri plasmid, respectively, must belinked to the genes to be integrated as a flanking region. Ifagrobacteria are used for the transformation, the DNA to be integratedmust be cloned into special plasmids, and specifically either into anintermediate or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid of the agrobacteria via homologousrecombination due to sequences that are homologous to sequences in theT-DNA. This plasmid also contains the vir-region, which is required forthe T-DNA transfer. Intermediate vectors cannot replicate inagrobacteria. A helper plasmid can be used to transfer the intermediatevector to Agrobacterium tumefaciens (conjugation). Binary vectors canreplicate both in E. coli and in agrobacteria. They contain a selectionmarker gene and a linker or polylinker, which are framed by the rightand left T-DNA border region. They can be transformed directly into thegrobacteria. The agrobacterial host cell should contain a plasmidcarrying a vir-region. The vir-region is necessary for transferring theT-DNA into the plant cell. Additional T-DNA can be present. Theagrobacterium transformed in this way is used to transform plant cells.The use of T-DNA for the transformation of plant cells has beenintensively studied and sufficiently described in generally knownarticles and manuals for plant transformation. Plant explants can bespecifically cultivated with Agrobacterium tumefaciens or Agrobacteriumrhizogenes for the transfer of DNA into the plant cells. Whole plantscan then be regenerated from the infected plant material (e.g., leafparts, stem segments, roots, but also protoplasts orsuspension-cultivated plant cells) in a suitable medium that can containantibiotics or biocides for the selection of transformed cells.

Once the introduced DNA has been integrated into the genome of the plantcell, it is generally stable there, and is maintained in the progeny ofthe originally transformed cell as well. It normally contains aselection marker, which makes the transformed plant cells resistant to abiocide or an antibiotic such as kanamycin, G 418, bleomycin,hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea,gentamycin or phosphinotricine, etc. Therefore, the individuallyselected marker should allow the selection of transformed cells fromcells lacking the introduced DNA. Also suited for this purpose arealternative markers, such as nutritive markers, screening markers (e.g.,GFP, green fluorescent protein). Naturally, selection markers need notbe used at all, although this would involve a fairly high screeningexpenditure. If marker-free transgenic plants are desired, the personskilled in the art also has strategies at his disposal that enablesubsequent removal of the marker gene, e.g., cotransformation,sequence-specific recombinases.

The transgenic plants are regenerated from transgenic plant cells byusual regeneration methods using known nutrient media. The plantsobtained in this way can then be analysed for the presence of theintroduced nucleic acid encoding a synthetic spider silk protein usingconventional methods, including molecular biological methods such as PCRand blot analyses.

The transgenic plant or transgenic plant cell can be any desiredmonocotyledonous or dicotyledonous plant or plant cell.

Useful plants or cells from useful plants are preferred. Especiallypreferred are transgenic plants selected from the group consisting ofthe tobacco plant (Nicotiana tabacum) and the potato plant (Solanumtuberosum).

The expression of the synthetic spider silk protein according to theinvention in the plants according to the invention or plant cellsaccording to the invention can be detected and followed usingconventional molecular biological and biochemical methods. The personskilled in the art knows these techniques and he can easily select asuitable detection method without any problem, e.g., a Northern blotanalysis or a Southern blot analysis.

FIG. 9 shows an example for the manufacture of transgenic spider silkprotein-producing plants. The PCR-amplified sequences can possiblycontain frame shift mutations. For this reason, the sequences accordingto the invention must be tested prior to the generation of transgenicplants. Performing a sequence analysis each starting from the flankingvector sequences can do this. Longer constructs of more than 1 kb cannotbe verified in this way, since due to the repetitive properties of theDNA sequences according to the invention internal sequencing primersprovide no reliable sequences that can be evaluated accurately. For thisreason, amplified spidroin sequences were preferably cloned into thebacterial expression vector pet23a (Novagen, Madison, USA). Byimmunodetection of the expression frame shift mutations may then beprecluded.

The nucleic acid molecules or expression cassettes according to theinvention are usually cloned as HindIII fragments into shuttle vectorssuch as pBIN, pCB301 and/or pGSGLUC1. These shuttle vectors arepreferably transformed in Agrobacterium tumefaciens. The transformationof Agrobacterium tumefaciens is usually verified via Southern blotanalysis and/or PCR screening.

The invention also relates to propagating material and harvest productsof the inventive plants, e.g., fruits, seeds, bulbs, tubers, seedlings,cuttings, etc.

Further, the invention relates to a method of obtaining plant spidersilk protein, comprising the following steps:

-   a) transfer of a recombinant nucleic acid molecule or vector    according to the invention containing a DNA sequence that codes for    a synthetic spider silk protein to plant cells;-   b) optionally, regeneration of plants from the transformed plant    cells;-   c) processing of the plant cells from a) or plants from b) to obtain    plant spider silk protein.

In another important aspect of this invention, methods of obtainingrecombinant manufactured spider silk proteins are provided that comprisethe transfer of an inventive recombinant nucleic acid molecule or vectorcontaining a DNA sequence that codes for a synthetic spider silk proteinto any cells, i.e. for example bacterial or animal cells in addition toplant cells. An essential characteristic of these methods according tothe invention is the purification step of the recombinantly manufacturedspider silk proteins, which among other things utilize the proteins'special properties vis-à-vis solubility when heated and/or when acid isadded.

In one embodiment of the method according to the invention, therecombinantly manufactured spider silk protein is purified byheat-treating the cell extract, e.g., a plant seed extract, andsubsequently separating the denatured proteins naturally occurring inthe cell, e.g. the native proteins of the plant, for example bycentrifugation. In this case, the beneficial feature of therecombinantly produced spider silk proteins is utilized, namely that theproteins maintain solubility when aqueous solutions are heated up toboiling point. In contrast, synthetic fibre proteins of the spider andsilkworm after expression in Pichia pastoris only remain in a dissolvedstatus when heated up to a temperature of 63° C., and then only for 10minutes.

In another embodiment of the method according to the invention ofobtaining recombinantly manufactured spider silk proteins, purificationis performed by adjusting an acidic pH by adding acid, preferablyhydrochloric acid, to the cell extract, for example to the plantextract.

The acidic pH, particularly a pH ranging from 1.0 to 4.0, morepreferably ranging from 2.5 to 3.5, most preferably a pH of 3.0, is heremaintained preferably for several minutes, more preferably for about 30minutes, at a temperature below room temperature, preferablyapproximately 4° C. Again, an unexpected property of the proteinsobtained by the method of the invention is exploited, namely that theyremain in solution during acidification specifically up to a pH of 3.0at 4° C. On the other hand the proteins naturally occurring in the cell,for example proteins that are produced naturally in the cell, areprecipitated by this treatment and are then separated, especially bycentrifugation.

The above-described solubility properties of the spider silk proteinsthat are recombinantly produced according to the invention are verysurprising, were not foreseeable in this form, and permit an efficient,fast and inexpensive purification procedure when extracted from cells,in particular plant cells.

In another embodiment of the method according to the invention, anucleic acid molecule that additionally comprises a nucleic acidsequence coding for ELPs is transferred to the cells. In this case thepurification of the recombinantly manufactured spider silk protein isperformed as follows: in a first step, the spider silk-ELP fusionprotein is enriched by heat-treating the crude extract. Surprisingly,the fusion proteins retain the excellent solubility of the spider silkproteins at high temperatures. The bulk of the proteins naturallyoccurring in the cells are precipitated during this temperatureincrease. In the next step, further increasing the temperature,preferably to a temperature of at least 60° C., precipitates the spidersilk-ELP fusion proteins. Precipitation preferably takes place in thepresence of a suitable salt concentration, e.g. a NaCl concentration ofat least 0.5 M, preferably in a range of from 1 M to 2 M. Finally, theELP fragment is cleaved, preferably via digestion with CNBr.

Through the method for obtaining recombinantly manufactured spider silkprotein according to the invention described above, the proteins inplants may be accumulated to high concentrations, preferably up to anexpression level of about 4% of the total soluble protein. Thus, for thefirst time, methods are provided that can be used for technicallyfeasible enrichment of recombinant spider silk protein.

In another aspect of the present invention, the spider silk proteinsaccording to the invention can be used to produce synthetic threads, aswell as films and membranes. Such products are especially suitable formedical applications, in particular for closing wounds and/or as framesor covers for artificial organs. Further, the films and membranes madeout of the spider silk proteins according to the invention can be usedas adhesion surfaces for cultivated cells, as well as for filteringpurposes.

This invention will be explained in the following examples, which servemerely to illustrate the invention, and are in no way to be understoodas restrictive.

EXAMPLES Example 1 Expression and Stable Accumulation of Synthetic FibreProteins of the Spider and Silkworm in the Endoplasmatic Reticulum ofLeaves or Tubers from Transgenic Tobacco and Potato Plants

FIGS. 10 a and b show the amino sequences of synthetic spider silkproteins having a high degree of homology with the spidroin 1 proteinfrom Nephila clavipes, the C-terminal and non-repetitive constant regionnot being shown. These synthetic spider silk proteins consist ofmodules, which in turn comprise successively arranged oligonucleotidesequences. The combination of several modules resulted in the assemblyof the various synthetic genes, wherein mixed forms with sequences basedon fibroin 1 have also been created.

Table 1 below lists various plant expression cassettes, which code forvarious synthetic fibre proteins according to the invention with thesequences SEQ ID NO: 30 to 40. TABLE 1 Calculated Plant Number of aminomolecular weight expression acids (with (with leader cassette leadersequence) sequence) Homology SB1 No. 1 - 149 AS 11 kDa spidroin 1 (SEQID No. 19) SD1 No. 2 - 182 AS 13 kDa spidroin 1 (SEQ ID No. 21) SA1 No.3 - 215 AS 16 kDa spidroin 1 (SEQ ID No. 26) SE1 No. 4 - 275 AS 20 kDaspidroin 1 (SEQ ID No. 20) SF1 No. 5 - 317 AS 24 kDa spidroin 1 (SEQ IDNo. 29) SM12 No. 6 - 410 AS 31 kDa spidroin 1 (SEQ ID No. 28) SO1 No.7 - 676 AS 52 kDa spidroin 1 (SEQ ID No. 27) SO1SM12 No. 8 - 1035 AS 82kDa spidroin 1 (SEQ ID No. 23) SO1SO1 No. 9 - 1301 AS 102 kDa  spidroin1 (SEQ ID No. 22) SO1SO1SO1 No. 10 - 1926 AS 151 kDa  spidroin 1 (SEQ IDNo. 24) FA2 No. 11 - 264 AS 20 kDa spidroin 1 (SEQ ID No. 25) andfibroin

The target-specific transport and accumulation of the sequencesaccording to the invention in the endoplasmatic reticulum of cells oftransgenic plants was achieved by an N-terminal signal peptide sequenceand a C-terminal ER retention sequence (KDEL, SEQ ID NO: 52). Adetection sequence in the form of a c-myc-tag at the C-terminal end ofthe transgenic synthetic fibre proteins permits the detection oftransgenic products in plant extracts.

Cassettes SO1 and FA2 are shown in detail as examples in FIGS. 10 a and10 b. The plant expression cassettes SB1, SD1, SA1, SE1, SF1, SM12,SO1SM12, SO1SO1 and SO1SO1SO1 were created according to the samestructural principle. Varying the basic module repeats results insynthetic fibre proteins containing a different number of amino acidsand correspondingly different molecular weight (see Table 1).

FIG. 2 describes schematically how the constructs mentioned above arearranged. The SmaI and NaeI restriction sites were introduced fordirectly cloning the synthetic fibre protein genes of the presentinvention. To this end, a PCR product containing the correspondingrestriction sites was cloned with the primer combination 5′-pRTRA-SmaIand 3′-pRTRA-NotI in the plasmid pRTRA ScFv SmaI/BamHIΔ via BamHI andNotI. Synthetic fibre protein genes were cloned from the fibre proteingene derivatives of plasmids 9905 or 9609 in vector pRTRA.7/3placeholder. Selection of restriction endonuclease recognition sequencesat the 5′- and 3′-end of the synthetic fibre protein genes (SmaI andNaeI) allows them to be freely combined with each other, and largerfibre protein genes can be assembled in one cloning step according tothe invention.

In this way, transgenic synthetic spider silk proteins were accumulatedto high concentrations in the endoplasmatic reticulum of transgenictobacco and potato plants (see FIGS. 12 a and 12 b). Table 2 shows themaximal accumulation level of synthetic spider silk proteins accordingto the invention in the ER of leaves of transgenic tobacco and potatoplants. The enrichment of transgenic synthetic fibre proteins wasestimated by means of a comparison with transgenic recombinantantibodies, which were likewise provided with the same tag. Thus for thefirst time, an accumulation of spider silk proteins in plants isdescribed using potato and tobacco as an example. TABLE 2 Fibre proteinSD1 SM12 SO1 FA2 Tobacco Accumulated amount in ˜0.5% ˜0.5% ˜0.5% ˜0.5%percentage of total protein Potato Accumulated amount in ˜0.5% ˜0.5%˜0.5% ˜0.5% percentage of total protein

A defined quantity of the fibre protein-containing total protein extract(40 μg) and a defined quantity of a reference protein withc-myc-immunotag (50 ng ScFv) were separated via SDS gel electrophoresis,and synthetic fibre proteins and reference proteins were detected in aWestern blot using an anti-c-myc antibody (see FIGS. 12 and 13). Thedata given as percentage values are derived from the comparison of theband intensity of the reference proteins and the band intensity of thesynthetic spider silk proteins according to the invention, and areestimated values. Differences in size of the synthetic fibre proteinsand reference protein were taken into account. Possible differences inlabelling efficiency can be almost precluded.

FIG. 13 shows the heat stability of various synthetic spider silkproteins according to the invention in plant extracts. Surprisingly, thespider silk proteins according to the invention remain in solution evenin a prolonged heat treatment of 3 hours (comparison of reference sampleR to samples H-60 min, H-120 min and H-180 min). More than 90% of theresidual plant proteins are denatured and can be simply separated outvia centrifugation (FIG. 13 a; comparison of sample R to H-60 min).These unusual properties of the synthetic spider silk proteins accordingto the invention, which among other things are a consequence of theiramino acid sequence and their folding in the plant ER, render possiblethe development of inexpensive purification strategies that can berealized on a large-scale.

FIG. 14 shows the solubility of synthetic fibre proteins from transgenicplants. In contrast to the bacterially expressed synthetic fibreproteins described in the prior art, the spider silk proteins accordingto the invention exhibit a surprisingly good solubility in aqueousbuffers (R1, R2=Tris buffer, T1, T2=phosphate buffer). These propertiesalso are attributable among other things to the amino acid sequence, andin particular the folding in the endoplasmatic reticulum of plant cells.

Example 2 Expression and Stable Accumulation of Synthetic Spider SilkProteins in the Cell Membrane of Leaves from Transgenic Tobacco andPotato Plants

This example describes the membrane-associated accumulation of spidersilk proteins according to the invention in transgenic tobacco andpotato plants. In this case, the constructs described in Example 1 thatare taken as the basis are used to produce fusion genes, which code foran spider silk protein and for a membrane domain. FIG. 15 shows ageneral diagram of these constructs. In this case, a NotI fragment wasisolated from the plasmid pRT-HOOK, which codes for both the HOOK domainand for a c-myc-immunotag, which then was cloned in spider silk proteingene-carrying derivatives of the pRTA.7/3 vector. Selection ofrestriction endonuclease recognition sequences at the 5′- and 3′-end ofthe synthetic spider silk protein genes (SmaI and NaeI) again allowsthem to be combined with each other in any order, so that larger fibreprotein genes can be assimilated in a single cloning step.

FIG. 16 shows the expression of the genes described above in transgenictobacco and potato plants. As can be seen from a comparison of samples1, 2 and 3 in this Figure, these transgenic spider silk proteins are notsoluble in the aqueous phase in contrast to the proteins according tothe invention described in Example 1. This property also can be utilizedfor the development of purification strategies.

Example 3 Targeted Alteration of the Solubility of Spider Silk Proteinsby Means of Fusion with Elastin-Like Peptides

In a first step it was shown that fusions with elastin-like peptidesalso result in an targeted alteration in the solubility behaviour as afunction of temperature and concentration even in spider silk proteinsexpressed in bacteria.

FIG. 5 shows a corresponding expression cassette. Examples for ELP with10, 20, 30, 40, 60, 70 and 100 pentameric units are identified in thesequences SEQ ID NO: 41 to 47. Examples for DNA sequences and amino acidsequences in the form of the construct SM12-70xELP as the plantexpression cassette or as the expression cassette for E. coli are shownin sequences SEQ ID NO: 48-51 or in FIGS. 19 to 22.

FIG. 17 shows the gel electrophoretic analysis of such a purificationtechnique. The spider silk-ELP fusion protein was enriched byheat-treating the crude extract. Surprisingly, the fusion proteinsretained the excellent solubility of the spider silk proteins at hightemperatures. The bulk of the E. coli proteins were precipitated out atthese temperatures.

After concentrating the enriched spider silk protein extract to a highlevel, the extract was subjected to a temperature of 60° C., after whichthe ELP spider silk protein precipitated and was removed via pelleting.The pellet was dissolved in water at room temperature, and insolublecomponents were removed via pelleting.

The spider silk protein fraction was then lyophilised and digested bycyanogen bromide cleavage. The cyanogen bromide cleavage was renderedpossible by the methionine residue between the spider silk protein andthe ELP peptide.

This was again followed by lyophilisation and dissolution in an aqueousbuffer. Concentration to a high level was then performed, wherein thecleaved ELP fragment (ELP(T-R); see FIG. 2) precipitated and was removedvia pelleting. The spider silk protein remained in solution (SM12(T-R);see FIG. 17). The solubility was maintained for a prolonged period, forSM12 at 4° C. for 24 h. The identity of spider silk protein purified inthis way was demonstrated by the peptide sequencing of the N-terminalend.

In a second step, spider silk proteins were accumulated as ELP fusionsin the endo-plasmatic reticulum of transgenic tobacco plants. FIG. 5also shows the basic structure of these expression cassettes. Thesefusion proteins having molecular weights of 35,000 Dalton to 100,000Dalton were all accumulated to high concentrations in plants with anexpression level of about 4% of the total soluble protein.

General Molecular Biological Methods

-   -   Cloning strategies: Restriction cleavages were performed in 100        μl end volume. As a standard, 10 μg of plasmid DNA, 10 Upper        restriction endonuclease, 10 μl of a suitable buffer (10×) were        used. DNA fragments were separated from each other via gel        electrophoresis, and purified by DNA gel extraction, where        necessary. For ligations, the DNA fragment (insert) to be cloned        was used in a threefold molar excess to the vector fragment.        Sticky-end ligations were performed in one hour, and blunt-end        ligations were performed in 12 h at 4° C. with 1 U ligase. The        DNA was incorporated both in the cells of E. coli and of A.        tumefaciens via electroporation. Transformants were selected on        suitable solid nutrient media with the addition of an antibiotic        (ampicillin or kanamycin).    -   PCR: PCR reactions were performed in 50 μl end volume. As a        standard, 100 ng of template DNA, 100 pmol of each primer, 1 μl        of dNTPs (10 mM) and 5 μl of a suitable buffer were used, along        with 1 U Tfl or Taq DNA polymerase. The following conditions        were selected for a PCR reaction: 2 min at 95° C., then 30        cycles, each running for 45 sec at 95° C., 45 sec at 50° C. or        55° C., 1 min at 72° C., followed by a cycle for 5 min at 72° C.    -   Expression and accumulation in tobacco and potato plants:        Transgenic plants were selected in an incubator room under        uniform illumination at about 20° C. on suitable solid nutrient        media containing antibiotic (kanamycin, rifampicin and        carbenicillin). After roots appeared, they were allowed to        continue growth in pots containing soil in a greenhouse.

As for the rest, the molecular biological and biochemical techniquesused in the resent invention can be looked up in available laboratorymanuals, e.g., in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, 2^(nd) edition, Cold Spring Harbour Laboratory Press, ColdSpring Harbour, New York.

FIGURES

FIG. 1:

Oligonucleotide sequences that code for spidroin-typical short aminoacid repeats.

FIG. 2:

Successive arrangement of oligonucleotide sequences for constructingmodules using the DNA sequences of the present invention.

FIG. 3:

Structure of DNA sequences according to the invention made out ofmodules.

FIG. 4:

Cloning of the gene of the HOOK transmembrane domain with NotI from(pRT-HOOK) in (pRTA.73 syn.spidroin).

FIG. 5:

Diagrammatic representation of the spidroin-ELP expression cassettes.xELP units: 10, 20, 30, 40, 60, 70 or 100 pentamers(Val-Pro-Gly-Val-Gly, SEQ ID NO: 54). The methionine between the spidersilk protein and the ELP peptide renders possible the cyanogen bromidecleavage.

FIG. 6:

Change of a base in the BamHI recognition sequence (position 1332) viatargeted mutagenesis.

FIG. 7:

Preparation of (pRTRA.73, BamHIA) for directly cloning the syntheticspidroin gene from p9905 or p9609—cancellation of the SmaI recognitionsequence (position 463).

FIG. 8:

Introduction of the restriction recognition sequences of SmaI and NaeIinto the vector (pRTRA.73, BamHIA+SmaIA) for cloning synthetic spidroingenes.

FIG. 9:

General depiction of the manufacture of transgenic plants producingspider silk protein.

FIG. 10:

(a) Depiction of the modular structure of the spider silk proteinsaccording to the invention based on the example of the SO1 sequence.Amino acids 1-28: LeB4 signal peptide; amino acids 29-659: syntheticspider silk protein sequence; amino acids 660-672: c-myc-tag; aminoacids 673-676: ER retention signal.

Arrangement of the sequence modules according to the original sequencespecified in Simmons et al., “Molecular orientation and two-componentnature of the crystalline fraction of spider dragline silk” (1996),Science 271: 84-87.

(b) Depiction of the modular structure of the synthetic fibre hybridprotein FA2. Amino acids 1-27: LeB4 signal peptide; amino acids 28-130:synthetic fibre protein sequence of the spider; amino acids 131-247:synthetic fibre protein sequence of the silkworm; amino acids 248-260:c-myc-tag; amino acids 261-264: ER retention signal.

FIG. 11:

Diagrammatic representation of the construction of gene cassettes forthe accumulation of synthetic fibre proteins of the spider and silkwormin the ER of transgenic plants.

FIG. 12:

(a) Expression of synthetic fibre proteins of the spider (SD 1, SM12,SO1) or the hybrid of spider and silkworm (FA2) in leaves of transgenictobacco plants. 40 μg of total protein were analysed in SDS samplebuffer. SD1: 13 kDa; FA2: 20 kDa; SM12: 31 kDa; SO1: 52 kDa; K: positivecontrol 50 ng ScFv.

(b) Expression of the synthetic fibre proteins of the spider (SD 1,SM12, SO1) or hybrid of spider and silkworm (FA2) in transgenic potatoplants.

40 μg of total protein were also analysed in the SDS sample buffer.SD1:1:3 kDa; FA2: 20 kDa; SM12: 31 kDa; SO1: 52 kDa; K: positive control50 ng ScFv.

FIG. 13:

Depiction of the heat resistance of the synthetic fibre proteins of thespider and silkworm based on the constructs SD1 and FA2. A:Coomassie-stained gel. B: Immunochemical detection of the syntheticfibre proteins SD1 and FA2 via anti-c-myc antibodies. PM: proteinmarker; ScFv: 50 ng ScFv; R: aqueous plant extract from leaves oftransgenic plants for SDI and FA2; H: heating step 60 min, 120 min, 180min, 24 h and 48 h at 90° C.

Plant extract constituents precipitated during heat treatment wereseparated by centrifugation.

FIG. 14:

Analysis of the solution properties and stability of the syntheticspider silk protein SO1 after ammonium sulfate precipitation.

10 g of leaf material were shock-frozen in liquid nitrogen, triturated,taken up in 20 ml of crude extract buffer, shaken for 30 min at 38° C.,and then insoluble components have been removed via centrifugation (30min, 10,000 rpm). The supernatant (R) was then heated to 90° C. for 10min, and the precipitate was removed via centrifugation (30 min, 10,000rpm). Ammonium sulfate saturated up to a concentration of 20% in thefinal volume was added to the supernatant (H), the mixture was stirredby rotation at room temperature for 4 h, and the precipitate was thenremoved via centrifugation for 60 min at 4000 rpm and 4° C. After thatammonium sulfate was added to the supernatant up to a concentration of30% saturation and the mixture was agitated overnight at roomtemperature. The solution was split into 5 aliquots, and the precipitatewas removed by centrifugation (60 min, 4000 rpm, 4° C.). Thesupernatants were discarded, and the remaining pellets were taken up inthe following solutions: R1: crude extract buffer (50 mM Tris/HCl pH8.0; 100 mM NaCl, 10 mM MgSO₄); S: SDS sample buffer; G: 0.1 M phosphatebuffer, 0.01 M Tris/HCl, 6 M guanidinium hydrochloride/HCl pH 6.5; T:1×PBS, 1% TritonX-100; L: LiBr.

The charges were shaken for 1 h at 37° C., and insoluble components wereremoved by centrifugation (30 min, 10,000 rpm). An aliquot of eachcharge was then removed in order to prepare SDS gel electrophoresis (R1,S1, G1, T1, L1). The charges were allowed to stand at room temperaturefor 36 h. Insoluble components were removed via centrifugation (30 min,10,000 rpm). An aliquot of each charge was again removed and preparedfor SDS gel electrophoresis (R2, S2, G2, T2, L2). Comparable volumeswere again analyzed.

FIG. 15:

Diagrammatic view of the construction of gene cassettes for theaccumulation of cell membraneous synthetic fibre proteins of the spiderand silkworm in transgenic plants.

FIG. 16:

Expression of the fibre fusion proteins SM12-HOOK, SO1-HOOK and FA2-HOOKin the leaves of transgenic potato plants.

FIG. 17:

Gel electrophoretic analysis of the enrichment of bacterially expressedspider silk proteins after fusion with ELPs. Spider silk protein: 30,000Dalton.

FIG. 18:

Western blot analysis of the expression of spider silk-ELP fusionproteins in transgenic tobacco plants. 2.5 μg of the total plant proteinwere separated, and the spider silk proteins were detected on theWestern blot by ECL. The spider silk protein concentration was estimatedto be at least 4% of the total soluble protein by comparing it with thestandard.

FIG. 19:

DNA sequence of SM12-70xELP as the plant expression cassette.

FIG. 20:

Protein sequence of SM12-70xELP from plant expression (SM12, c-myc-tag,70xELP, KDEL (SEQ ID NO: 52)—depicted in that order).

FIG. 21:

DNA sequence of SM12-70xELP as expression cassette for E. coli.

FIG. 22:

Protein sequence of SM12-70xELP from bacterial expression (SM12,c-myc-tag, 70xELP, c-myc-tag, HisTag—depicted in that order).

1-37. (canceled)
 38. A DNA sequence that codes for a synthetic spidersilk protein having a homology of at least 80% to the spidroin 1 proteinof Nephila clavipes, wherein the DNA sequence comprises modulescomprising a group of successively arranged oligonucleotide sequences,wherein the oligonucleotide sequences each code for repetitive unitsfrom spidroin proteins, wherein the modules are freely arranged, andwherein the oligonucleotide sequences are selected from the groupconsisting of: a) TATGAGCGCTCCCGGGCAGGGT (SEQ ID NO: 1); b)AGCTTTTAGGTACCAATATTAATCTGGCCGGCTCCACC (SEQ ID NO: 2); c) TATGGTCTGGGG(SEQ ID NO: 3); d) GGCCAGGGTGCTGGCCAA (SEQ ID NO: 4); e)GGTGCAGGAGCWGCWGCWGCWGCTGCAGGTGGA (SEQ ID NO: 5); f)GCCGGCCAGATTAATATTGGTACCTAAA (SEQ ID NO: 6); g) CTGCCCGGGAGCGCTCA (SEQID NO: 7); h) ACCACCATAACCTCC (SEQ ID NO: 8); i) AGCACCCTGGCCCCCCAG (SEQID NO: 9); j) TGCAGCWGCWGCWGCWGCTCCTGCACCTTGGCC (SEQ ID NO: 10); k)TATGAGATCTGGCCAAGGAGGT (SEQ ID NO: 11); l) TTGGCCAGATCTCA (SEQ ID NO:12); m) AGTCAGGGTGCTGGTCGTGGAGGCCAA (SEQ ID NO: 13); n)TCCACGACCAGCACCCTGACTCCCCAG (SEQ ID NO: 14); o)AGTCAGGGCGCTGGTCGTGGGGGACTGGGTGGCCAA (SEQ ID NO: 15); p)ACCCAGTCCCCCACGACCAGCGCCCTGACTCCCCAG (SEQ ID NO: 16); q)CTGGGAGGGCAGGGAGCGGGCCAA (SEQ ID NO: 17); r) CGCTCCCTGCCCTCCCAGACCTCC(SEQ ID NO: 18); and s) sequences that exhibit at least 80% sequenceidentity to the sequences a) to r).
 39. The DNA sequence according toclaim 38, wherein the modules comprise at least 4 oligonucleotidesequences.
 40. The DNA sequence according to claim 38, wherein the DNAsequence comprises at least 4 modules.
 41. The DNA sequence according toclaim 38, wherein the DNA sequence further comprises nucleic acidsequences that code for repetitive units from fibroin proteins.
 42. TheDNA sequence according to claim 38 comprising one of the sequencesidentified in SEQ ID NO. 19 to
 29. 43. A recombinant nucleic acidmolecule comprising a) a DNA sequence that codes for a synthetic spidersilk protein wherein the DNA sequence comprises modules comprising agroup of successively arranged oligonucleotide sequences, wherein theoligonucleotide sequences each code for repetitive units from spidroinproteins, wherein the modules are freely arranged such that it ispossible for the synthetic spider silk protein to exhibit an alteredrange of properties in comparison to native spider silk protein, b) anubiquitously acting promoter, and c) a nucleic acid sequence coding forELPs.
 44. The recombinant nucleic acid molecule according to claim 43,wherein the DNA sequence is a DNA sequence according to claim
 38. 45.The recombinant nucleic acid molecule according to claim 43 furthercomprising at least one nucleic acid sequence that codes for a plantsignal peptide.
 46. The recombinant nucleic acid molecule according toclaim 45, wherein the plant signal peptide mediates the transport intothe endoplasmatic reticulum (ER).
 47. The recombinant nucleic acidmolecule according to claim 45, wherein the nucleic acid sequence thatcodes for the plant signal peptide is an LeB4Sp sequence.
 48. Therecombinant nucleic acid molecule according to claim 43 furthercomprising a nucleic acid sequence that codes for an ER retentionpeptide.
 49. The recombinant nucleic acid molecule according to claim11, wherein the ER retention peptide comprises the KDEL sequence (SEQ IDNO: 52).
 50. The recombinant nucleic acid molecule according to claim 43further comprising a nucleic acid sequence that codes for atransmembrane domain.
 51. The recombinant nucleic acid moleculeaccording to claim 50, wherein the nucleic acid sequence codes for thetransmembrane domain of the PDGF receptor.
 52. The recombinant nucleicacid molecule according to claim 43, wherein the ELPs comprise from 10to 100 pentameric units.
 53. The recombinant nucleic acid moleculeaccording to claim 43 comprising one of the sequences identified in SEQID NO. 48 and
 50. 54. A microorganism comprising a recombinant nucleicacid molecule according to claim
 43. 55. A recombinant spider silkprotein coded by a DNA sequence according to claim
 38. 56. The spidersilk protein according to claim 55, wherein the protein's molecularweight ranges from 10 to 160 kDa.
 57. A recombinant spider silk proteincomprising one of the amino acid sequences identified in SEQ ID NO. 30to
 40. 58. A method of manufacturing spider silk protein-producingplants or plant cells comprising the following steps: a) manufacturing arecombinant nucleic acid molecule according to claim 43; b) transferringthe nucleic acid molecule from a) to plant cells; and c) optionally,regenerating plants from the transformed plant cells.
 59. Transgenicplant cells comprising a recombinant nucleic acid molecule according toclaim
 43. 60. Transgenic plants comprising a plant cell according toclaim 59 as well as parts of these plants, transgenic harvest productsand transgenic propagating material of these plants, such asprotoplasts, plant cells, calli, seeds, tubers, cuttings, and thetransgenic progeny of these plants.
 61. The transgenic plants accordingto claim 60 selected from the group consisting of tobacco plants andpotato plants.
 62. A method of obtaining plant spider silk proteincomprising the following steps: a) transferring a recombinant nucleicacid molecule according to claim 43 to plant cells; b) optionally,regenerating plants from the transformed plant cells; and c) processingthe plant cells from a) or plants from b) to obtain plant spider silkprotein.
 63. A method of obtaining recombinant manufactured spider silkprotein comprising the following steps: a) transferring a recombinantnucleic acid molecule according to claim 43 to cells; and b) purifyingthe spider silk protein by heat-treating the cell extract and thenseparating the denatured proteins naturally occurring in the cell.
 64. Amethod of obtaining recombinant manufactured spider silk proteincomprising the following steps: a) transferring a recombinant nucleicacid molecule according to claim 43 to cells; and b) purifying thespider silk protein by adjusting an acidic pH by adding acid to the cellextract and then separating the denatured proteins naturally occurringin the cell.
 65. The method according to claim 64, wherein the acidic pHranges from 2.5 to 3.5.
 66. The method according to claim 64, whereinthe acid is hydrochloric acid.
 67. A method of obtaining recombinantmanufactured spider silk protein comprising the following steps: a)transferring a recombinant nucleic acid molecule according to claim 43to cells; and b) purifying the spider silk protein as follows: i)enriching the spider silk-ELP fusion protein by heat-treating the cellextract; ii) precipitating the spider silk-ELP fusion protein by furtherincreasing the temperature; and iii) cleaving off the ELP fragment. 68.The method of claim 67, wherein the temperature is at least 60° C. 69.The method of claim 67, wherein the ELP fragment is cleaved off viadigestion with CNBr.
 70. The method according to claim 63, wherein thecells are selected from the group consisting of plant cells, animalcells and bacterial cells.
 71. The method according to claim 64, whereinthe cells are selected from the group consisting of plant cells, animalcells and bacterial cells.
 72. The method according to claim 67, whereinthe cells are selected from the group consisting of plant cells, animalcells and bacterial cells.
 73. A method of manufacturing syntheticthreads, films and/or membranes from spider silk proteins derived from aDNA sequence according to claim
 38. 74. The method according to claim73, wherein the threads, films and/or membranes are used for medicalpurposes.
 75. The method according to claim 74, wherein the threads,films and/or membranes are used for closing wounds and/or as frames orcovers for artificial organs.
 76. The method according to claim 73,wherein the films and/or membranes are used as adhesion surfaces forcultivated cells and/or for filtering purposes.
 77. The DNA sequenceaccording to claim 38, wherein at least one property of the syntheticspider silk protein selected from the group consisting of tensilestrength, elasticity, swelling capacity, solubility behaviour, acidstability and heat resistance is altered compared to a native spidersilk protein.